1. Field of the Invention
The invention relates to a device which can sort particles (sample) such as cells and blood corpuscles on the basis of scattering angle information of scatter.
2. Related Art
Flow cytometers, which is a one of kind of particle analyzer, include a cell sorter, a cell analyzer, and a particle analyzer (blood analyzer) which incorporate these devices. Such a flow cytometer is configured in the following manner. Particles flowing through or dropping from a flow cell are irradiated with light. Forward scatter, orthogonal scatter, or the like which is produced as a result of the irradiation is detected. Particles such as cells and blood corpuscles are sorted on the basis of the detected optical information.
It is known that, in forward scatter, the scattering angle varies depending on the kind of particles. Therefore, a device is proposed in which forward scatter is collected with being split into plural regions of different scattering angles, and forward scatter of each region is measured, thereby sorting particles. An example of such a device is disclosed in Unexamined Japanese patent publication HEI. 8-271509. In the device, cells contained leukocytes are optically detected so that cells of a small content are accurately sorted. FIGS. 8 and 9 show the configuration for detecting forward scatter in the disclosed device.
As shown in FIG. 9 including a section view of a flow cell used in the flow cytometer, the interior of the flow cell 2 has a structure wherein a sample tube 2c into which the sample (particles) flows is positioned in a center portion of a sheath fluid 2a which flows in a sheath-like manner and a sample flow 2b containing particles are formed in the tip end portion of the tube so as to be surrounded by the sheath fluid, thereby continuously supplying particles to a capillary portion. Since the capillary portion is irradiated with laser light a, a particle 7 passing through the portion function as the scattering source to produce forward scatters b, c, and d, orthogonal scatters e, f, and g (these scatters are directed to the back face of the sheet, and hence not illustrated), and back scatters h and i.
As shown in FIG. 8, the laser light emitted from the light source 1 is transmitted through the flow cell 2 and then blocked by an obscurator 11 which is immovably disposed. The forward scatter b, c, d or the like is converted into a collimated pencil of ray by a collimator lens 3 which is disposed in front of the flow cell 2, and then impinges on a forward-disposed mirror 4. A hole 4a is formed at the center of the mirror 4. Light of a small scattering angle in the forward scatter passes through the hole 4a and is then collected onto a first detector 6 by a first collecting lens 5. Light of a large scattering angle in the forward scatter impinges on the periphery of the mirror 4 to be reflected thereby so that the light pathway is changed by 90 deg., and is then collected onto a second detector 6xe2x80x2 by a second collecting lens 5xe2x80x2. Each of the first and second detectors 6 and 6xe2x80x2 is configured by a light/voltage converter element such as a photodiode, and converts received light into an electric signal corresponding to the intensity of the light. The signal is supplied to an analyzer 9.
In other words, the first detector 6 detects the intensity of forward small angle scatter having a small scattering angle, and the second detector 6xe2x80x2 detects that of forward large angle scatter having a large scattering angle. The analyzer 9 calculates forward small angle scatter data and forward large angle scatter data from the incoming detection signals, and stores the calculated data. The analyzer calculates also 2-dimension coordinate data of the intensities of forward small angle scatter and forward large angle scatter. The analyzer 9 sequentially supplies the calculated forward small angle scatter data and forward large angle scatter data to an external display device (not shown) so that the data are displayed on a screen as a scattergram of a 2-dimension coordinate.
It is known that scatters produced by laser light irradiation have the following directional properties:
when the size of a particle is larger than the wavelength of the laser light, forward scatter is produced;
when the size of a particle is at a similar degree as the wavelength of the laser light, forward and orthogonal scatters are produced; and
when the size of a particle is smaller than the wavelength of the laser light, forward, orthogonal, and back scatters are produced.
In order to sort particles more correctly, therefore, an actual device is configured in the following manner. As shown in FIG. 9, for the forward scatters b, c, and d, an optical system 8 having a collimator lens 3, perforated mirrors 4, and collecting lenses 5, and detectors 6 for detecting the intensities of the scatters are disposed; for the orthogonal scatters e, f, and g also, an optical system 8 and detectors 6 are disposed; and, for back scatters h and i also, an optical system 8 and detectors 6 are disposed.
FIGS. 10(a)-10(b) shows another device described in Unexamined Japanese patent application HEI. 8-271509. In the device, light is not split into plural regions of different scattering angles, but split by using an optical fiber bundle 10. Namely, forward scatter emitted from particles in the flow cell 2 impinges on a light receiving face of the optical fiber bundle 10. In the light receiving face, as shown in FIG. 10(b), an optical fiber group 10a for receiving forward small angle scatter is disposed at the center, and an optical fiber group 10b for receiving forward large angle scatter is disposed in the periphery. As shown in FIG. 10(a), the emission side of the optical fiber group 10a is connected to the first detector 6, and that of the optical fiber group 10b to the second detector 6xe2x80x2. According to this configuration, scattering light entering the light receiving face of the optical fiber bundle 10 is supplied to either of the detectors 6 and 6xe2x80x2 by a predetermined optical fiber group, in accordance with the scattering angle. In the same manner as the device of FIG. 8, the outputs of the first and second detectors 6 and 6xe2x80x2 are supplied to the analyzer and then undergo a predetermined signal process.
However, the above-mentioned devices of the prior art have the following problems. In the device shown in FIG. 8, the optical for enabling the detectors 6 and 6xe2x80x2 to receive light requires the collimator lens 3, the perforated mirror 4, and the collecting lenses 5. Therefore, the number of parts is increased, and the adjustment of the optical system is complicated and cumbersome. Furthermore, the optical system occupies a large area, and hence miniaturization of the whole of the device is impeded. In a device such as shown in FIG. 9 in which the number of split regions is increased and orthogonal scatters and back scatters are to be further detected, perforated mirrors, the number of which is equal to (the division number xe2x88x921), and collecting lens the number of which is equal to the division numbers, are required. This causes the number of parts to be further increased, with the result that the above-mentioned problems become more serious.
In the device shown in FIGS. 10(a)-10(b), the collimator lens and the collecting lenses are not required, and the optical system requires only the optical fiber bundle. Even when the number of split regions is increased, it is necessary to increase only the number of optical fiber bundles. Unlike the device of FIGS. 8 and 9, the increase of the part number, and that of the occupied area are not large. However, the light receiving faces of optical fiber bundles must be concentrically arranged, and optical fibers must be collected in the unit of bundle. Therefore, a cumbersome process of routing optical fibers must be conducted. This causes the division number to be limited. Furthermore, the use of optical fibers brings a large loss. Specifically, a loss is produced when light passes through optical fibers, and light impinging on a clad is not transmitted. As a result, 50% or less of light impinging on the light receiving face of optical fiber bundle 10 shown in FIG. 10(a) can finally reach the detectors, thereby lowering the use efficiency of light.
The invention has been conducted in view of the above-mentioned circumstances. It is an object of the invention to provide a particle analyzer which can solve the problems discussed above, in which light can be split into plural regions of different scattering angles by a simple configuration, an optical system can be easily adjusted, and the use efficiency of light is high, in which, even when the number of split regions is increased, the number of parts of an optical system for splitting light is not increased so that the occupied area is not increased, and only an increase of the space due to an increased number of detectors is required, and in which the posture of a composite lens constituting the main portion of the invention can be performed easily and correctly so as to attain high measurement accuracy.
In order to attain the object, the particle analyzer of the invention is a particle analyzer in which particles are sorted by separately detecting regions of forward scatter which is produced by irradiating the particles with light, the regions having different scattering angles, and configured so that the particle analyzer comprises: a composite lens which is placed so as to block a light pathway of the forward scatter, and in which plural ring-like lens elements of different focal positions are integrally joined together in a state in which the lens elements are concentrically positioned, and a positioning element is optionally disposed in a center area; plural detectors for different scattering angles (in an embodiment, second and third detectors 17 and 18), the detectors being respectively placed at imaging positions of the forward scatter, the imaging positions respectively corresponding to the focal positions of the plural lens elements; a positioning detector (in the embodiment, a first detector 16) which can detect light which has passed through the positioning element; and analyzing means for sorting the particles on the basis of detection outputs of the plural detectors, and an optical axis of the positioning element coincides with at least a center of the composite lens, and, when the composite lens is perpendicular to the optical axis, the positioning element allows a predetermined amount of light to be received at a predetermined position of the positioning detector.
The imaging positions respectively corresponding to the focal positions where the detectors are disposed are not always coincident with the focal positions because of the following reason. The scatter impinging on the composite lens is not a parallel beam, and hence an image is not formed at the focal position. However, each of the imaging positions can be uniquely decided, because scattering occurs at a position of particles flowing through or dropping from the flow cell and the relative positional relationship between the flow cell and the composite lens is known.
According to this configuration, forward scatter impinges on a predetermined position of the composite lens. Specifically, forward small angle scatter having a small scattering angle impinges on a ring-like lens element which is positioned in a center area of the composite lens, and forward large angle scatter having a large scattering angle impinges on a lens element which is positioned in the periphery of the composite lens. Since the lens elements have different focal positions, the scatters impinging on the lens elements are formed into images at different positions, and then detected by the detectors, respectively.
In the invention, the positioning element is disposed at the center of the composite lens. Therefore, judgement on whether the posture of the composite lens attains a desired state or not can be easily performed by monitoring the output of the positioning detector. As a result, the positioning and the posture control can be easily performed.
The predetermined amount of light means an amount of light which is larger than a given reference. The given reference includes the following cases: when a pinhole is used as the positioning element, the given reference may be a predetermined value which is larger than 0; and, when a photodiode having a pinhole is used as the detector and a convex lens is used as the positioning element, for example, the reference value is 0 and it is judged whether the amount of light is larger than 0 or not (light is received or not).
Various elements may be used as the positioning element For example, a convex lens may be used, or a pinhole may be used.
The composite lens may be a conventional lens. When the composite lens is configured by a fresnel lens, the flow cytometer can be easily designed and produced, and the handling is facilitated. Therefore, this configuration is preferable.
The structure for attaining different focal points may be realized by various manners. For example, plural lens elements of different focal lengths may be joined together, plural lens elements of different principal axes may be joined together, or these structures may be combined with each other.
Boundary portions of the plural lens elements, and/or those of the periphery of the positioning element and the lens elements may be covered by annular masking members, respectively. When such portions are covered by masking members, light collection is performed on the basis of light passing through portions of the lens elements where the composition is stable. Consequently, noise components hardly enter the signals output from the detectors, and a measurement can be performed in a further accurate manner.
Furthermore, an obscurating member which blocks direct light emitted from a light source may be disposed at a predetermined position between a flow cell to which the particles are supplied, and the composite lens, and the obscurating member may be retractable when the composite lens is to be adjusted by using the positioning element. During a measurement, when direct light from the light source which is larger in amount and forward scatter impinge simultaneously on the detector, information of the forward scatter is canceled. To comply with this, usually, a flow cytometer is configured so that an obscurating member is disposed at a predetermined position so as to prevent direct light from impinging on a composite lens. When the posture of the composite lens is to be adjusted, the positioning element at the center of the composite lens must be irradiated with light. If such an obscurating member is disposed, however, it is impossible to irradiate the positioning element with light. Therefore the, obscurating member may be configured so as to be retractable, and, when the composite lens is to be adjusted, the obscurating member may be retracted so that the positioning element is irradiated with light, thereby allowing both the positioning and the measurement to be surely performed.
This retraction may be realized by, for example, a configuration in which the obscurating member is mounted on a slide-rail or the like and the member is moved along the slide-rail. In this case, the member may be coupled to driving means such as a motor so that the retraction is automatically performed. Alternatively, the obscurating member may be manually removed away from the disposition position. An example of such manual removal will be described. The obscurating member is detachably mounted on a holder for the composite lens. When the positioning is to be performed, the member is detached from the holder, and, when the measurement is to be performed, the member is attached to the holder.
In the invention, the positioning element is placed in the light pathway of direct light. When direct light spreads in a degree smaller than the positioning element, therefore, the lens elements are not irradiated with direct light and forward scatter. As a result, even when the intensity of direct light is high, the obscurating member is not always required.